1. Field of Invention
This invention relates to a method and apparatus that allows filters with absorbed bacterial or mammalian cell samples to be analyzed for specific genes and gene products. More particularly this invention relates to a device that allows the experimenter to perform sequential operations or analysis on one or a large number of filters to identify bacterial or mammalian cells which contain specific genes.
2. Prior Art
Protein-encoding DNA of different organisms can be inserted into various strains of bacteria, creating what is commonly known as a cDNA Library. Generally, enzymes duplicate the mRNA of an organism into thousands of individual complimentary DNA fragments. These DNA fragments are then packaged in certain phages, which, by well known biomechanisms, insert the DNA segments into the genome of a particular strain of bacteria.
The bacterial colonies are cultivated on agar plates. The bacterial colony which contains the gene of interest is detected by a variation of replica plating. Nitrocellulose filters are applied to the surface of the colonized agar plates. Bacteria adhere to the nitrocellulose filters or gene products secreted by bacteria are absorbed into the filter. Reference marks on the filter paper and the plate make it possible to run various assays on the filter paper and later identify the colony, if any, which harbors the particular gene of interest. As thousands to literally millions of individual bacterial colonies are cultivated, each containing a small fragment of the specimen genome, ten to one hundred of agar plates may be needed to grow the colonies. Consequently, this same number of filters would be used to sample the colonies on each agar plate.
The colony containing the gene of interest can be identified by radiolabelled DNA or mRNA probes. The gene product can be traced by using monoclonal or polyclonal antibodies coupled with enzymatic or fluorescent tags, or by means of enzymatic assays. Other protein to protein or protein to DNA interactions can be detected in a similar way.
A new technique which uses phage technology to introduce complementary DNA for antibody heavy and light chain fragments into strains of E.Coli will substantially increase the need for rapid sequential analysis of large numbers of filters. The bacteria or phage that produce antibody fragments are cultured on agar plates. A filter is laid on top of the agar plate to absorb the antibody fragments. These filters are then tested against solutions containing labelled antigen. This technique is essentially the reverse of the method previously described.
Whatever detection method is chosen, each one of the sample filters must be subjected to the same assay method under controlled conditions. These tests require that the filters be agitated in various solutions which contain the reagents for the assay and then washed in buffer solutions.
One method of agitating filters in solution is to place the filters in a heat sealed or lock plastic bag of the type frequently used in kitchens. A solution is added to the bag, the bag is sealed, and the filters are agitated inside the bag by hand or simply allowed to soak.
This method has several limitations. The most severe of which is the uneven application of the antibody or reagent solution to the filter surface and diffusion through the filter. This problem is caused by air bubbles in the plastic bag, contact between the sides of the bag and the filter surface, contact between the surfaces of two or more filters in the bag or limited diffusion through a stack of filters. Effective washing of the filters with buffers and other solutions is similarly retarded.
Other disadvantages are also present. The filters are more likely to tear or otherwise be damaged during the agitation process. Bubbles can form and be trapped between filters. The size of the bag limits the number of filters that can be reacted or washed at one time. It is often difficult to control various experimental parameters using a plastic bag such as reaction time, temperature and reagent concentration. Further, the plastic bag method results in an unacceptable number of false positive reactions.
In a second method, the filters are placed in glass crystallizing dishes which contain the reaction or wash solution. The dishes are covered with a lid or a plastic wrap. The dish is then placed on a rotating platform which agitates the filter inside the dish. The disadvantages of this technique are myriad. If several filters are placed in a crystallizing dish, they must frequently be separated because of a tendency to stick to one another and to the sides of the dish. In addition, a separate crystallizing plate must be used for each step of the reaction or wash. During the transfer from one dish to the next, the filter may become contaminated or torn and wash or incubation liquid is easily spilled. Evaporation of the reaction or wash solution may adversely effect the concentration of the reactants. Finally, with a sequential analysis using several such dishes, it is difficult to control such variables as time, temperature and concentration when transferring the filter from dish to dish.
A device described by Larry W. Cohen in Bio Techniques, Vol. 8, No. 4 (1990) employs two plexiglass boxes to apply solutions containing radiolabelled probes to filters. One box is filled with a radioactive solution. A smaller box has contiguous groves cut in two sides and the bottom. Hybridization filters are placed in the grooves. The box holding the filters is then lowered into the box containing the solution. When the reaction is finished, the smaller box is raised and drained.
There are several disadvantages connected with this device. First, the groove system provides insufficient support for filters immersed in solution causing the filters to collapse against each other and the wall of the vessel when the filter box is removed. Because there is no method of draining the larger box, the filter box must be removed and the larger box filled and drained by hand. This makes it difficult to control parameters of temperature and concentration. In addition, the filters may be contaminated or dry out when exposed to air. Further, in order to do a series of sequential analyses, several boxes would have to be used, each containing a different reactor or wash solution. Although agitation would facilitate the reaction and washing of the filters, no means for agitating the filters was apparent.
Other patents describe devices and processes for sequential analysis of bacterial samples. U.S. Pat. No. 4,237,096 describes a device which is a series of reaction chambers. Liquid containing whole bacteria, lysed bacteria or secreted gene products is introduced into the chamber by a gravity feed channel. The liquid flows into each chamber by means of a ball valve. When the desired amount of sample is in each chamber, a reagent is added to the sample chambers. Plainly, the usefulness of this device is limited to testing of samples in liquid media. Further, the analyses must produce color change reaction to be detectable. Finally, because the samples cannot be washed, probes such as labelled monoclonal or polyclonal antibodies could not be used.
U.S. Pat. No. 4,632,901 describes a method and apparatus for doing immunoassays. The device has two members the first of which is a filter with a monoclonal antibody bound to it. A second member is made up of an absorbent material. A liquid sample is poured onto the test filter through a funnel device which houses the filter. The absorbent material facilitates the flow of liquid through the filter. Antigens in the sample are bound to the antibody in the filter. Radio labelled antibody is added, followed by several washes. The filter is then tested for the presence of labelled antibody. The main disadvantage of this device is that only one filter can be tested at a time. Further, this process is not suitable for sequential analysis. Finally, reactions that require controlled parameters such as reactant concentration, temperature, and reaction time cannot be done with this device.
U.S. Pat. No. 4,673,638 describes a method for detecting microorganisms which produce a desired substance. A porous membrane of inert material is placed on top of the agar surface in a growth plate. Bacteria are grown on the membrane. Secreted substances pass through the membrane into the agar. The bacteria can also be lysed allowing non-secreted substances to run through the membrane into the agar. The membrane with the growing bacteria is lifted from the agar surface and stored appropriately. Reagents in the agar itself, or placed on the surface of the agar, react with the bacterial substrate and a reaction is observed in the agar. Although it is claimed that more colonies can be screened by this method it is clear that only one analysis can be run on each agar plate. In addition, it would not be practical to use radio or flourescene labelled probes because of background problems created by diffiusion of the probe solution into the agar.
Therefore, it is an object of this invention to provide a reaction-wash vessel for use with various sizes and types of filters.
It is another object of this invention to provide a reaction-wash vessel that allows individual filters to be easily inserted and removed.
It is another object of this invention to provide a reaction-wash vessel that allows up to 100 filters to be reacted and washed at one time.
It is still a further object of this invention to provide a reaction-wash vessel that allows filters to be agitated without adhering to the side of the apparatus or to other filters.
It is a still further object of this invention to provide a reaction-wash vessel that provides for uniform application of a reagent and/or wash solution to the surface of single or multiple filters.
It is still a further object of this invention to provide a reaction-wash vessel that allows multiple filters to be assayed under similar controlled conditions.
It is still a further object of this invention to provide a reaction-wash vessel in which several different analyses and washes can be done on sample filters in the same reaction-wash vessel.
It is still a further object of this invention to provide a reaction-wash vessel in which bubbles are hindered from forming and adhering to the surface of a filter during a reaction or wash.
It is still a further object of this invention to provide a means for precisely controlling the temperature of the reaction-wash vessel filter solution.
It is still a further object of this invention to provide a process for performing sequential analysis on multiple sample filters.